Abstract 196: Self-Organized Formation of Cardiac Pacemaker Tissues in Embryoid Bodies

The pacemaker tissues of the heart are a complex set of specialized cells that initiate the rhythmic heart beat. The sinoatrial node (SAN) serves as the primary pacemaker, whereas the atrioventricular node (AVN) serves as a subsidiary pacemaker under conditions of SAN failure. The elucidation of genetic networks regulating the development of these tissues is crucial for understanding the mechanisms underlying arrhythmias and for the design of biological pacemakers. At present, there is no in vitro model system in which these specialized cells are defined or targeted. Here we report efficient self-organized formation of the two pacemaker nodes in three-dimensional aggregate cultures of mouse embryonic stem (ES) cells known as embryoid bodies (EBs), as well as the isolation of ES cell-derived AVN cells. A Shox2-lacZ knockin ES cell line carrying a Cx30.2 enhancer-RFP construct was used to generate EBs. The Shox2 gene, a determinant of the SAN genetic cascade, was used to delineate the SAN in EBs. The Cx30.2 enhancer was used to direct RFP expression to the AVN in EBs. Using live fluorescent imaging and immunohistochemistry, we demonstrate that these genetic markers reproducibly delineate cell clusters which express nodal proteins. These clusters are functionally connected with, and consistently located adjacent to the contracting region of the EB in an organized manner. We demonstrate that EBs generated using Shox2 knockout ES cells exhibit a hypoplastic SAN phenotype that includes reduction in spontaneous contraction rates and altered expression of Shox2 downstream targets. We isolated an ES cell-derived AVN cell line using a genetic selection technique and demonstrated that these cells display nodal characteristics such as calcium dependent spontaneous depolarizations and an AVN gene expression profile. When these cells were grown as three-dimensional aggregates they induced synchronous contraction of surrounding cardiac myocytes in co-culture experiments. Using molecular markers, we have generated a reproducible model system and have isolated an AVN cell line that will be invaluable tools for studying the molecular pathways regulating the development of the cardiac pacemaker tissues and molecular composition of these specialized cells.